Alkaline Bismuth Solution as an En Bloc Stain for Formaldehyde-Glutaraldehyde Potassium Permanganate Fixed Fungal Spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

Aldehyde Pararosanilin (True Aldehyde Fuchsin) Stains Purified Insulin, Oxidized or Not, Following Adequate Fixation with Either Formalin or Bouin'S Fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from “basic fuchsin” whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC.

Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content.

We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam® sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

Aldehyde pararosanilin (true aldehyde fuchsin) stains purified insulin, oxidized or not, following adequate fixation with either formalin or Bouin's fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from "basic fuchsin" whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC. Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content. We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

Alkaline Bismuth Stain as A Tracer for Golgi Vesicles of Plant Cells

An alkaline solution of bismuth subnitrate reacts well with carbohydrate-rich components of Golgi bodies in sections prepared from plant leaves fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. The metal deposits formed are so fine that the stain is appropriate to ultrastructural observation at high magnification. The Golgi vesicles show polarity with respect to the localization of the reactive deposits. Golgi vesicles that had migrated farther from the Golgi cisternae showed greater reactive deposits and higher membrane contrast than those close to the Golgi cisternae. These results indicate that the alkaline bismuth stain is an excellent tracer for Golgi bodies of plant cells.     

Lead asparate, an en bloc contrast stain particularly useful for ultrastructural enzymology

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.     

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Klig-man's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.     

LANTHANUM STAINING OF THE SURFACE COAT OF CELLS : Its Enhancement by the Use of Fixatives Containing Alcian Blue or Cetylpyridinium Chloride

Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex.     

An investigation of aldehyde fuchsin staining of unoxidized insulin

Aldehyde fuchsin is a standard stain for the secretion granules of pancreatic B cells. The participation of either insulin or proinsulin in aldehyde fuchsin staining is in dispute. There is some evidence that permanganate oxidized insulin is stained by aldehyde fuchsin. Aldehyde fuchsin staining of unoxidized insulin has not been investigated adequately despite excellent staining results with tissue sections. Unoxidized insulin and proinsulin suspended by electrophoresis in polyacrylamide gels were fixed with Bouin's fluid and placed in aldehyde fuchsin for one hour. Because the unoxidized proteins were not stained by aldehyde fuchsin, it was concluded that neither insulin or proinsulin are responsible for the intense aldehyde fuchsin staining of unoxidized pancreatic B cell granules in tissue sections. A series of controlled experiments was undertaken to test the effects of fixatives, oxidation and destaining procedures on aldehyde fuchsin staining of insulin, proinsulin and other proteins immobilized in polyacrylamide gels. It was demonstrated that only oxidized proteins were stained by aldehyde fuchsin and that cystine content of the proteins had no apparent relation to aldehyde fuchsin staining. It was concluded that neither insulin nor proinsulin is likely to be responsible for the intense aldehyde fuchsin staining of unoxidized pancreatic B cell granules in tissue sections.     

THE PARASPORAL BODY OF BACILLUS LATEROSPORUS LAUBACH

On sporulation the slender vegetative rods swell and form larger spindle-shaped cells in which the spores are formed. When the spores mature they lie in a lateral position cradled in canoe-shaped parasporal bodies which are highly basophilic and can be differentiated from the surrounding vegetative cell cytoplasm with dilute basic dyes. On completion of sporulation the vegetative cell protoplasm and the cell wall lyse, leaving the spore cradled in its parasporal body. This attachment continues indefinitely on the usual culture medium and even persists after the spores have germinated. In thin sections of sporing cells the bodies are differentiated from the cell protoplasm by differences in structure. Whereas the protoplasm has a granular appearance, in both longitudinal and cross-sections the parasporal body comprises electron-dense lamellae running parallel with the membranes of the spore coat and less electron-dense material in the interstices of the lamellae. The inner surface of the body is contiguous with that of the spore coat as if it were part of the spore, rather than a separate body attached to the spore. The staining reactions of the parasporal body are not consistent with those of any substance described in bacteria. With Giemsa the bodies stain like chromatin, but the Feulgen reaction indicates that they do not contain the requisite nucleic acid. With an aqueous solution of toluidine blue they stain metachromatically, but with an acidified solution the results are variable. Neisser's stain for polyphosphate is negative. The basophilic substance is removed from the body with some organic solvents. This basophilic substance has not been specifically identified with any material seen in ultrathin sections, but it is suggested that it might be the less electron-dense material in the interstices of the lamellar structure. In contrast to the spore coat of B. laterosporus, those of its two relatives B. brevis and B. circulans take up basic stain like the parasporal body. Thin spore sections of these species have shown that the walls are thicker than those surrounding the spores of B. laterosporus, and it is suggested that the outer stainable layer of brevis and circulans spores is an accessory coat which in laterosporus may have been deformed to give a parasporal body.     

Keratin in the Egg Shells of Amphistomes; Histochemical Differentiation from Quinonetanned Protein in Other Trematoda

Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H₂SO₄ for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H₂SO₄ and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.     

Identification of cells by backscattered electron imaging of silver stained bulk tissues in scanning electron microscopy

Anuran tadpole tail muscle was stained en bloc by a modified light microscope silver stain for light microscopy and freeze-fractured in liquid nitrogen after partial dehydration with ethanol. The fractured specimens were observed in both secondary electron and backscattered electron modes in a scanning electron microscope. Since the cell nuclei specifically stained with silver provided high contrast against the unstained background due to atomic number contrast of backscattered electron image, various cells were easily identified by a comparison of secondary electron images and compositional images of backscattered electron signals.     

Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.     

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

Fluorescein isothiocyanate-labeled β-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.     

Ultrastructure of Tobacco Ringspot Virus-Induced Local Lesions in Lima Bean Leaves

Lignin is hypothesized to be a factor in the ability of cucurbits to resist fungal infection. To determine if lignin was present around sites of infection, susceptible cucumber plants and cucumber plants with systemic induced resistance were inoculated with Colletotrichum lagenarium, and the ultrastructure of plant and pathogen was examined 24 to 72 hours later. Conidia produced germ tubes with appressoria within 24 hours of inoculation on both control and induced plants. Penetration of plant epidermal cell walls occurred by 48 hours, and papillae formed at the site of penetration. Both elemental bromine and potassium permanganate were used to histochemically stain for lignin. Potassium permanganate, used together with energy dispersive X-ray microanalysis (EDS), indicated the presence of lignin in papillae and in electron-dense areas of plant cell walls directly beneath the appressoria. Using EDS, silicon was shown to be localized in the electron-dense areas of cucumber leaf cells. This is the first report of silicon in induced cucumber plants. There were no qualitative differences between the resistance reactions of induced and control plants.     

Optimal preparation of mycelia and arthrospores of Geotrichum candidum: a SEM, TEM and cytochemical study

Three morphological stages of Geotrichum candidum , viz. the mycelium, a cylindrical spore type, and an ellipsoidal spore type, were subjected to qualitative and quantitative morphological and cytochemical analyses using scanning and transmission electron microscopy. Considerable variation in total cell shrinkage as well as cell wall thickness was found with fixation procedure and cell type. Cell walls were best preserved with simultaneous fixation in aldehydes and osmium whereas optimal preservation of the cytoplasm required sequential fixation. Mechanical pretreatment improved the visualization of ultrastructural details in the thick–walled ellipsoidal spores. Wall polysaccharides and cytoplasmic glycogen stained positively with periodic acid–thiosemicarbohydrazide–silver proteinate and with alkaline bismuth subni–trate. In all three cell types an electron–transparent wall region was sandwiched between outer and inner electron–dense and PAS–positive regions. Wall thickness in cylindrical spores corresponded to that of mycelium whereas ellipsoidal spores had a 1.7 times thicker wall and a 1.9 times greater wall volume than cylindrical spores.     

Vacuole Partitioning during Meiotic Division in Yeast

We have examined the partitioning of the yeast vacuole during meiotic division. In pulse-chase experiments, vacuoles labeled with the lumenal ade2 fluorophore or the membrane-specific dye FM 4-64 were not inherited by haploid spores. Instead, these fluorescent markers were excluded from spores and trapped between the spore cell walls and the ascus. Serial optical sections using a confocal microscope confirmed that spores did not inherit detectable amounts of fluorescently labeled vacuoles. Moreover, indirect immunofluorescence studies established that an endogenous vacuolar membrane protein, alkaline phosphatase, and a soluable vacuolar protease, carboxypeptidase Y, were also detected outside spores after meiotic division. Spores that did not inherit ade2- or FM 4-64-labeled vacuoles did generate an organelle that could be visualized by subsequent staining with vacuole-specific fluorophores. These data contrast with genetic evidence that a soluble vacuolar protease is inherited by spores. When the partitioning of both types of markers was examined in sporulating cultures, the vacuolar protease activity was inherited by spores while fluorescently labeled vacuoles were largely excluded from spores. Our results indicate that the majority of the diploid vacuole, both soluble contents and membrane-bound components, are excluded from spores formed during meiotic division.     

Selective staining of fungal hyphae in parasitic and symbiotic plant-fungus associations

A cytochemical method for light microscopical studies is described which allows the specific detection of fungal hyphae in plant-fungus associations: e.g. lichens, mycorrhiza, or fungal infections of plant tissue. The specimens were fixed and embedded in epoxy resin by a standard protocol for electron microscopy. Semithin sections were successively incubated with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and calcofluor white (CW). FITC-WGA stained exclusively the fungal cell walls while CW stained both the fungal and the plant cell walls. Therefore, FITC-WGA is an excellent marker for the fungal hyphae.     

Staining of Fungi With Saccharated Iron Oxide

A 0.2% aqueous solution of saccharated iron oxide with a pH of 10.8 is shown to be suitable for supravital staining of fungi. Colonies or fresh tissue materials presumed to contain fungi are immersed in the solution for 1-24 hr, fixed in a 10% neutral solution of buffered formalin about 1 hr, washed, and treated with 10% potassium ferrocyanide in 0.5 N, HC1. Cell membranes of hyphae, perithekia, and walls of sporangia appear dark blue; cytoplasm of the hyphae, sporangiospores and spores stain greenish blue or yellowish green.     

Staining of xylem parenchyma mitochondria with photo-oxidized 3,3'-diaminobenzidine

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.     

Staining of Xylem Parenchyma Mitochondria with Photo-Oxidized 3,3'-Diaminobenzidine

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldebyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.